Adhesion molecules from the diatom Phaeodactylum tricornutum (Bacillariophyceae): genomic identification by amino‐acid profiling and in vivo analysis

Cell adhesion molecules (CAMs) are important in prokaryotes and eukaryotes for cell–cell and cell–substratum interactions. The characteristics of adhesive proteins in the model diatom Phaeodactylum tricornutum were investigated by bioinformatic analysis and in vivo characterization. Bioinformatic analysis of the protein coding potential of the P. tricornutum genome used an amino‐acid profile that we developed as a new system to identify uncharacterized or novel CAMs. Putative diatom CAMs were identified and seven were characterized in vivo, by generation of transgenic diatom lines overexpressing genes encoding C‐terminal yellow fluorescent protein (YFP) fusion proteins. Three of these selected genes encode proteins with weak similarity to characterized proteins, a c‐type lectin and two fasciclins, whereas the others are novel. The resultant cell lines were investigated for alterations in their adhesive ability. Whole cell‐substratum adhesion strength was measured in a fully turbulent flow chamber, while atomic force microscopy was used to quantify the relative frequency of adhesion, as well as the length and strength of single molecules in the secreted mucilage. Finally, quartz crystal microbalance analysis characterized the visco‐elastic properties and interaction of the mucilage–substratum interface. These combined studies revealed a range of phenotypes affecting adhesion, and led to the identification of candidate proteins involved in diatom adhesion. In summary, our study has for the first time combined bioinformatics and molecular physiological studies to provide new insights into diatom adhesive molecules.     

Extracellular matrix assembly in diatoms (Bacillariophyceae). iv. ultrastructure of Achnanthes longipes and Cymbella cistula as revealed by high-pressure freezing/freeze substituton and cryo-field emission scanning electron microscopy

Extracellular matrix (ECM) polymers secreted by the diatoms Achnanthes longipes Ag. and Cymbella cistula (Ehr.) Kirchn. completely encase the cell and are responsible for adhesion and other interactions with the external environment. To preserve details of the highly hydrophilic ECM in the native state and to preserve, with a high degree of fidelity, the intracellular structures involved in synthesis of extracellular polymers, we applied a suite of cryotechniques. The methods included high‐resolution visualization of surfaces using cryo‐field emission SEM (cryo‐FESEM) and preservation for TEM observation of thin sections by high‐pressure freezing (HPF) and freeze substitution (FS). The extracellular structures of diatoms plunge‐frozen in liquid ethane, etched at low temperature, and observed on a cryostage in the FESEM showed overall dimensions and shapes closely comparable to those observed with light microscopy. Cryo‐FESEM demonstrated the pervasive nature of the extracellular polymers and their importance in cell–substratum and cell–cell associations and revealed details of cell attachment processes not visible using other SEM techniques or light microscopy. The layer of ECM coating the frustule and entirely encapsulating cells of A. longipes and C. cistula was shown to have a significant role in initial cell adhesion and subsequent interaction with the environment. Trails of raphe‐associated ECM, generated during cell motility, were shown at high resolution and consist of anastomoses of coiled and linear strands. Cryo‐FESEM revealed a sheet‐like mucilage covering stalks. HPF/FS of A. longipes resulted in excellent preservation of intra‐ and extracellular structures comparable to previous reports for animals and higher plants and revealed several organelles not described previously. Three distinct vesicle types were identified, including a class closely associated with Golgi bodies and postulated to participate in formation of the extracellular adhesive structures. HPF/FS showed a number of continuous diatotepic layers positioned between the plasma membrane and the silicon frustule and revealed that extracellular adhesive extrusion through frustule pores during stalk production was closely related to the diatotepum. The stalks of A. longipes consist of highly organized, multilayered, fine fibrillar materials with an electron‐opaque layer organized as a sheath at the stalk periphery.     

EXTRACELLULAR MATRIX ASSEMBLY IN DIATOMS (BACILLARIOPHYCEAE). V. ENVIRONMENTAL EFFECTS ON POLYSACCHARIDE SYNTHESIS IN THE MODEL DIATOM,PHAEODACTYLUM TRICORNUTUM1

The effects of phosphate (P) limitation, varying salinity (5–65 psu), and solid media growth conditions on the polysaccharides produced by the model diatom, Phaeodactylum tricornutum Bohlin were determined. Sequential extraction was used to separate polymers into colloidal (CL), colloidal extracellular polymeric substances (cEPS), hot water soluble (HW), hot bicarbonate soluble (HB), and hot alkali (HA) soluble fractions. Media‐soluble polymers (CL and cEPS) were enriched in 4‐linked mannosyl, glucosyl, and galactosyl residues as well as terminal and 3‐linked xylosyl residues, whereas HW polymers consisted mainly of 3‐linked glucosyl as well as terminal and 2,4‐linked glucuronosyl residues. The HB fraction was enriched in terminal and 2‐linked rhamnosyl residues derived from the mucilage coating solubilized by this treatment. Hot alkali treatment resulted in the complete dissolution of the frustule releasing 2,3‐ and 3‐linked mannosyl residues. The fusiform morphotype predominated in standard and P‐limited cultures and cultures subjected to salinity variations, but growth on solid media resulted in an enrichment of the oval morphotype. The proportion and linkages of 15 residues, including neutral, uronic acid, and O‐methylated sugars, varied with environmental conditions. P limitation and salinity changes resulted in 1.5‐ to 2.5–fold increase in carbohydrate production, with enrichment of highly branched/substituted and terminal rhamnose, xylose, and fucose as well as O‐methylated sugars, uronic acids, and sulfate. The increased deoxy‐ and O‐methylated sugar content under unfavorable environments enhances the hydrophobicity of the polymers, whereas the anionic components may play important roles in ionic cross‐linking, suggesting that these changes could ameliorate the effects of salinity or P‐stress and that these altered polysaccharide characteristics may be useful as bioindicators for environmental stress.     

Ongoing speciation within the Anastrepha fraterculus cryptic species complex: the case of the Andean morphotype

The Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae) cryptic species complex is currently composed of seven taxonomically recognized morphotypes. Both, pre‐ and post‐zygotic isolation has been documented among four of these morphotypes, revealing that in fact they appear to be distinct biological entities. In order to progress in the full delimitation of species within the complex, we examined reproductive isolation between a Colombian population of the Andean morphotype and populations belonging to four other morphotypes spanning from Mexico to Argentina. Flies from the Andean morphotype exhibited strong pre‐zygotic mating isolation through temporal partitioning of mating activity. Post‐zygotic isolation was observed for crosses of males of all morphotypes and Andean morphotype females, yet most of the F1 hybrid ♂ × F1 hybrid ♀ self‐crosses showed normal levels of fertility, a finding suggesting a nuclear–cytoplasmic interaction according to previous studies. Overall, the Andean morphotype within the complex also appears to be a distinct biological entity. We discuss the implications of these findings for the understanding of speciation mechanisms in the Neotropical genus Anastrepha.     

THE STRUCTURE AND NANOMECHANICAL PROPERTIES OF THE ADHESIVE MUCILAGE THAT MEDIATES DIATOM‐SUBSTRATUM ADHESION AND MOTILITY1

We investigated the adhesive mucilage and mechanism of cell‐substratum adhesion of two benthic raphid diatoms, the marine species Craspedostauros australis E. J. Cox and the freshwater species Pinnularia viridis (Nitzsch) Ehrenberg. SEM images of P. viridis and C. australis cells revealed the presence of multistranded tethers that appear to arise along the raphe openings and extend for a considerable distance from the cell before forming a “holdfast‐like” attachment with the substratum. We propose that the tethers result from the elongation/stretching of composite adhesive mucilage strands secreted from raphes during the onset of cell adhesion and reorientation. Atomic force microscopy (AFM) force measurements reveal that the adhesive strands originating from the nondriving raphe of live C. australis and P. viridis are highly extensible and accumulate to form tethers. During force measurements tethers can be chemically stained and are seen to extend between the cantilever tip and a cell during elongation and relaxation. In most cases, AFM force measurements recorded an interaction with a number of adhesive strands that are secreted from the raphe. The force curves of C. australis and P. viridis revealed a sawtooth pattern, suggesting the successive unbinding of modular domains when the adhesive strands were placed under stress. In addition, we applied the “fly‐fishing” technique that allowed the cantilever, suspended a distance above the cell, to interact with single adhesive strands protruding from the raphe. These force curves revealed sawtooth patterns, although the binding forces recorded were in the range for single molecule interactions.     

THE COMPLEX POLYSACCHARIDES OF THE RAPHID DIATOM PINNULARIA VIRIDIS (BACILLARIOPHYCEAE)1

A combination of carbohydrate analysis and atomic force microscopy (AFM) was used to characterize the polysaccharides of the pennate diatom, Pinnularia viridis (Nitzsch) Ehrenberg. Polymeric substances were fractionated into those in the spent culture medium (SCM) and those sequentially extracted from the cells with water at 45° C (WW), NaHCO₃ containing EDTA at 95° C (HB), and 1 M NaOH containing NaBH₄ at 95° C. Carbohydrate, protein, and sulfate were detected in all the fractions, but their relative proportions differed significantly. Nineteen sugars were identified, including pentoses, hexoses, 6‐deoxyhexoses, O‐methylated sugars, aminohexoses, and traces of uronic acids. To some extent, the same constituent monosaccharides and a proportion of the linkage patterns occurred in all four fractions, indicating the fractions contained a spectrum of highly heterogeneous but structurally related polysaccharides. Several carbohydrates were enriched in specific fractions. A soluble, partially substituted, 3‐linked galactan was slightly enriched in the SCM. The WW fraction was highly enriched in 3‐linked glucan, presumably derived from chrysolaminaran. Chemical and AFM data for the WW and HB fractions indicated that compositional differences were associated with substantial changes in the morphology and properties of the cell surface mucilage. Soluble polymers relatively enriched in fucose conferred a degree of softness and compressibility to the mucilage, whereas most of the mucilage comprised firmer more gelatinous polymers comparatively enriched in rhamnose. The frustule residue dissolved during extraction with NaOH, and a partially substituted 3‐linked mannan, together with relatively large amounts of protein, was obtained.     

Identification of the maternal source of young‐of‐the‐year Arctic charr in Lake Hazen,Canada

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The role of COBRA‐LIKE 2 function,as part of the complex network of interacting pathways regulating Arabidopsis seed mucilage polysaccharide matrix organization

The production of hydrophilic mucilage along the course of seed coat epidermal cell differentiation is a common adaptation in angiosperms. Previous studies have identified COBRA‐LIKE 2 (COBL2), a member of the COBRA‐LIKE gene family, as a novel component required for crystalline cellulose deposition in seed coat epidermal cells. In recent years, Arabidopsis seed coat epidermal cells (SCEs), also called mucilage secretory cells, have emerged as a powerful model system for the study of plant cell wall components biosynthesis, secretion, assembly and de muro modification. Despite accumulating data, the molecular mechanism of COBL function remains largely unknown. In the current research, we utilized genetic interactions to study the role of COBL2 as part of the protein network required for seed mucilage production. Using correlative phenotyping of structural and biochemical characteristics, unique features of the cobl2 extruded mucilage are revealed, including: ‘unraveled’ ray morphology, loss of primary cell wall ‘pyramidal’ organization, reduced Ruthenium red staining intensity of the adherent mucilage layer, and increased levels of the monosaccharides arabinose and galactose. Examination of the cobl2cesa5 double mutant provides insight into the interface between COBL function and cellulose deposition. Additionally, genetic interactions between cobl2 and fei1fei2 as well as between each of these mutants to mucilage‐modified 2 (mum2) suggest that COBL2 functions independently of the FEI‐SOS pathway. Altogether, the presented data place COBL2 within the complex protein network required for cell wall deposition in the context of seed mucilage and introduce new methodology expending the seed mucilage phenotyping toolbox.     

Substratum adhesion and gliding in a diatom are mediated by extracellular proteoglycans

Diatoms are unicellular microalgae encased in a siliceous cell wall, or frustule. Pennate diatoms, which possess bilateral symmetry, attach to the substratum at a slit in the frustule called the raphe. These diatoms not only adhere, but glide across surfaces whilst maintaining their attachment, secreting a sticky mucilage that forms a trail behind the gliding cells. We have raised monoclonal antibodies to the major cell surface proteoglycans of the marine raphid diatom Stauroneis decipiens Hustedt. The antibody StF.H4 binds to the cell surface, in the raphe and to adhesive trails and inhibits the ability of living diatoms to adhere to the substratum and to glide. Moreover, StF.H4 binds to a periodate-insensitive epitope on four frustule-associated proteoglycans (relative molecular masses 87, 112, and >200 kDa). Another monoclonal antibody, StF.D5, binds to a carbohydrate epitope on the same set of proteoglycans, although the antibody binds only to the outer surface of the frustule and does not inhibit cell motility and adhesion. Received: 2 December 1996 / Accepted: 6 March 1997     

Adhesive modular proteins occur in the extracellular mucilage of the motile, pennate diatom Phaeodactylum tricornutum

This Letter reports on adhesive modular proteins recorded by atomic force microscopy on live cells from the extracellular mucilage secreted from, and deposited around, the motile form of the pennate diatom Phaeodactylum tricornutum. This is the first report of modular proteins and their supramolecular assemblies, called adhesive nanofibers (ANFs), to be found on diatoms that use adhesives not only for substratum adhesion, but as a conduit for cell motility. The permanent adhesive pads secreted by Toxarium undulatum, a sessile centric diatom, were previously shown to possess ANFs with a modular protein backbone. Our results reported here suggest that modular proteins may be an important component of diatom adhesives in general, and that diatoms utilize the tensile strength, toughness, and flexibility of ANFs for multiple functions. Significantly, the genome of P. tricornutum has recently been sequenced; this will allow directed searches of the genome to be made for genes with modular protein homologs, and subsequent detailed studies of their molecular structure and function.     

THE POLYMORPHIC DIATOM PHAEODACTYLUM TRICORNUTUM: ULTRASTRUCTURE OF ITS MORPHOTYPES1,2

The ultrastructure of the oval, fusiform and triradiate morphotypes of Phaeodactylum tricornutum Bohlin is described. The organization and structure of the cytoplasmic organelles is similar in all three morphotypes, except that the vacuoles occupy the extra volume created by the arms of the fusiform and triradiate cells. The frustule in fusiform and triradiate cells is organic; in the oval type it may be organic or one of the valves may have a silica frustule surrounded by an organic wall. In all cells, the organic cell wall has up to 10 silica bands (13 nm wide) embedded in its surface in the girdle region, lacks girdle bands, and has an outer corrugated cell wall layer, except in the girdle region. Cell division, organic wall formation and silica deposition are described in detail. Four types of oval cells are also described. The relation to other diatoms is discussed.     

Nanostructure and nanomechanics of live Phaeodactylum tricornutum morphotypes

The ultrastructure and mechanical properties of the fusiform, triradiate and ovoid morphotypes of Phaeodactylum tricornutum were investigated using atomic force microscopy. Using topographic imaging, we showed that the surface of the ovoid form is rougher than those of the two other specimens, and coated with an outer layer of extracellular polymers. Using spatially resolved force–indentation curves, we found that the valve of the ovoid form is about five times stiffer (Young modulus of ∼500 kPa) than those of the other forms (∼100 kPa), a finding fully consistent with the fact that only the ovoid form has a silica valve, whereas the valves in the other two consist mostly of organic material. Notably, the girdle region of both fusiform and ovoid forms was five times softer than the valve, suggesting that this region is poor in silica and enriched in organic material. For the triradiate form, we showed the arms to be softer than the core region, presumably as a result of organelle localization. Last, we observed mucilaginous footprints of moderate stiffness (∼100 kPa) in the vicinity of ovoid diatoms, which we believe are secreted extracellular polymers.     

To be or not to be planktonic? Self‐inhibition of biofilm development

The transition between planktonic growth and biofilm formation represents a tightly regulated developmental shift that has substantial impact on cell fate. Here, we highlight different mechanisms through which bacteria limit their own biofilm development. The mechanisms involved in these self‐inhibition processes include: (i) regulation by secreted small molecules, which govern intricate signalling cascades that eventually decrease biofilm development, (ii) extracellular polysaccharides capable of modifying the physicochemical properties of the substratum and (iii) extracellular DNA that masks an adhesive structure. These mechanisms, which rely on substances produced by the bacterium and released into the extracellular milieu, suggest regulation at the communal level. In addition, we provide specific examples of environmental cues (e.g. blue light or glucose level) that trigger a cellular response reducing biofilm development. All together, we describe a diverse array of mechanisms underlying self‐inhibition of biofilm development in different bacteria and discuss possible advantages of these processes.     

Bacteria may induce the secretion of mucin‐like proteins by the diatom Phaeodactylum tricornutum

Benthic diatoms live in photoautotrophic/heterotrophic biofilm communities embedded in a matrix of secreted extracellular polymeric substances. Closely associated bacteria influence their growth, aggregation, and secretion of exopolymers. We have studied a diatom/bacteria model community, in which a marine Roseobacter strain is able to grow with secreted diatom exopolymers as a sole source of carbon. The strain influences the aggregation of Phaeodactylum tricornutum by inducing a morphotypic transition from planktonic, fusiform cells to benthic, oval cells. Analysis of the extracellular soluble proteome of P. tricornutum in the presence and absence of bacteria revealed constitutively expressed newly identified proteins with mucin‐like domains that appear to be typical for extracellular diatom proteins. In contrast to mucins, the proline‐, serine‐, threonine‐rich (PST) domains in these proteins were also found in combination with protease‐, glucosidase‐ and leucine‐rich repeat‐domains. Bioinformatic functional predictions indicate that several of these newly identified diatom‐specific proteins may be involved in algal defense, intercellular signaling, and aggregation.     

Morphology and molecular phylogeny of coccoid green algae Coelastrella sensu lato (Scenedesmaceae,Sphaeropeales), including the description of three new species and two new varieties

The genus Coelastrella was established by Chodat (Bull. Soc. Bot. Geneve, 13 [1922] 66), and was characterized as being unicellular or in few‐celled aggregations with many longitudinal ribs on the cell wall. Many species of this genus showed strong ability to accumulate carotenoids and oils, so they have recently attracted much attention from researchers due to its potential applicability in the energy and food industries. In this study, a total of 23 strains of Coelastrella were sampled from China, and three new species and two new varieties were described: C. thermophila sp. nov., C. yingshanensis sp. nov., C. tenuitheca sp. nov., C. thermophila var. globulina var. nov., C. rubescens var. oocystiformis var. nov. Besides 18S rDNA and ITS2 sequences, we have newly sequenced the tufA gene marker for this taxon. Phylogenetic analysis combined with morphological studies revealed four morphotypes within the Coelastrella sensu lato clade, which contained the morphotype of original Coelastrella, original Scotiellopsis, Asterarcys, and morphotype of C. vacuolata and C. tenuitheca sp. nov. The relationships between morphological differences and phylogenic diversity based on different markers were discussed. Our results support that 18S rDNA was too conserved to be used a species‐specific or even a genus‐specific marker in this clade. The topology of tufA gene‐based phylogenetic tree had a better match with the morphological findings.     

Antigenic variation in mucilage secreted by members of the genus Symbiodinium (Dinophyceae)

Symbiodinium reside intracellularly in a complex symbiosome (host and symbiont‐derived) within cnidarian hosts in a specific host‐symbiont association. Symbiodinium is a diverse genus with variation greater than other dinoflagellate orders. In this paper, our investigation into specificity examines antigenic variation in the algal mucilage secretions at the host‐symbiont interface. Cultured Symbiodinium from a variety of clades were labeled with one of two antibodies to symbiont mucilage (PC3, developed using a clade B alga cultured from Aiptasia pallida; BF10, developed using a clade F alga cultured from Briareum sp.). The labeling was visualized with a fluorescent marker and examined with epifluorescence and confocal microscopy. PC3 antigen was found in cultured Symbiodinium from clades A and B, but not clades C, D, E and F. The correlation between labeling and clade may account for some of the specificity between host and symbiont in the field. Within clades A and B there was variation in the amount of label present. BF10 antigen was more specific and only found in cultures of the same cp23S‐rDNA strain the antibody was created against. These results indicate that the mucilage secretions do vary both qualitatively and quantitatively amongst Symbiodinium strains. Since the mucilage forms the host‐symbiont interface, variation in its molecular composition is likely to be the source of any signals involved in recognition and specificity.     

The structure of anoline (Reptilia: Dactyloidae: Anolis) toe pads in relation to substratum conformity

Adhesive toe pads of geckos house modified components of vascular and/or connective tissues that promote conformity of the setal fields with the locomotor substratum. Similar modifications have been claimed for the digits of Anolis, but evidence for them is not compelling. Angiographic and histological investigations of Anolis failed to identify any evidence of either an intralamellar vascular reticular network or a central sinus. Instead, their vascularity more closely resembles that of lizards in general than that of pad‐bearing geckos. The loose connective tissue of the toe pads likely contributes to their general pliability and flexibility, promoting localized compliance with the substratum. Through the shedding cycle, the lamellae change shape as the replacing setae elongate. The outer epidermal generation lacunar cells on the inner lamellar faces simultaneously hypertrophy, providing for compatibility between overlapping lamellae, enabling reciprocity between them. This contributes to continuing compliance of the setal fields with the substratum. Overall, digital structure and attachment and release kinematics of the toe pads of Anolis are very similar to those of geckos exhibiting an incipient adhesive mechanism. Both lack major anatomical specializations for promoting conformity of the setae with the locomotor substratum beyond those of the seta‐bearing portions of the epidermis.     

Coprolites from shallow marine deposits of the Nanjemoy Formation,Lower Eocene of Virginia,USA

The Eocene Nanjemoy Formation crops out on the Maryland and Virginia Coastal Plain, along the eastern coast of the United States. This formation is composed of sands, silts and clays and is divided into the Potapaco and Woodstock members. Remains of fishes, reptiles, birds, mammals, molluscs, fruits and seeds are common in the Potapaco Member, in addition to vertebrate coprolites. Here, we present an analysis of more than 2000 coprolites from the Fisher/Sullivan Site in Virginia. The chemical composition (phosphatic) and the type of inclusions (fish bones) indicate that only scats of carnivorous animals were preserved. The analysed specimens were grouped into six morphotypes: (1) the cylindrical morphotype is a cylinder with rounded ends; (2) the segmented morphotype is a cylinder segmented with rounded ends, and occasionally one end is concave; (3) the oval morphotype represents a bean‐shaped coprolite; (4) the scroll morphotype is cylindrical to conical in lateral view and has coils seen only at the ends; (5) the folded morphotype is a spiral that is concentrically folded; and (6) the sinuous morphotype is serpentine, with rounded ends. Coprophagy‐related scrape traces occur in different morphotypes and represent both invertebrate burrows and bite traces made by fishes. The mineralogical and chemical analyses indicate an early precipitation of phosphate and pyrite minerals, probably induced by the microbial community. All coprolites at the Fisher/Sullivan Site were produced by fishes: carcharhiniform sharks for the scroll morphotype and lamniform sharks, probably the genus Carcharias, for the folded morphotype; the oval, cylindrical and segmented morphotypes were likely produced by actinopterygian fishes.     

ACTIVE GLIDING MOTILITY IN AN ARAPHID MARINE DIATOM,ARDISSONEA (FORMERLY SYNEDRA) CRYSTALLINA1

Active gliding movement over long distances was observed and filmed in the marine pennate diatom Ardissonea (Synedra) crystallina (Agardh) Kütz. Typical speeds measured ca. 1–2 μm-s^?1. Motion wax often smooth and steady; however, discontinuous jerky motions and rolling movements were common. Motion, was associated with secretion of twin or, less commonly, single straight trails of mucilage from one end of the cell. In a few instances, reversal in direction was related to cessation of mucilage secretion at one end and commencement at the other. Temporary cessation of movement due to an obstruction was accompanied by a build-up of mucilage at one end of the cell. Mucilage was apparently secreted at two specific sites at each end of the cell and was stained by alcian blue. Persistent trails were visible under scanning electron microscopy (SEM). SEM confirmed that cells had no raphes or labiate processes. The apparent site of secretion was a deep groove formed at the junction of the valve and valvocopula (first girdle band) at each end of the cell. Transmission electron microscopy confirmed the presence of mucilage vesicles in the cytoplasm, but these were not in any manner obviously related to secretion nor was any morphological structure associated with secretion. Cells often become epiphytic through secretion of a terminal stipe. Both stipe secretion and movement may involve the same structural differentiation of the frustule. These results demonstrate a previously unrecorded type of diatom motility. The mechanism, involves mucilage secretion and appears similar to that seen, for example, in some other algae such as the desmids (green algae).     

Nucleoids and coated vesicles of “Epulopiscium” spp.

We describe here aspects of the anatomy of two “Epulopiscium” morphotypes, unusually large bacteria that are not yet cultured and that reproduce by the internal generation of two or more vegetative daughter cells. Two morphotypes, A and B, which are enteric symbionts of several species of herbivorous surgeonfish (Acanthuridae), were collected around the Great Barrier Reef of Australia, preserved there, and later stained for light microscopy. Some samples were examined by electron microscopy. In both morphotypes, countless discrete nucleoplasms or nucleoids were found to occupy a single shallow layer just beneath the surface all around these organisms. At each end of the morphotype B cells, a membrane-bound compartment containing dense cords of chromatin was observed. When these were found at each end of growing daughter cells, no polar compartments were then found in their mother organism. Electron micrographs of sections of morphotype A symbionts show that their outermost region is composed of tightly packed coated vesicles, each surrounded by a thin, dense, spacious capsule. Near the surface of type A organisms the remains of broken vesicles, broken capsules, and a finely fibrous matrix fuse to form a fabric that serves as the cell wall. Morphotype B organisms, however, were observed to have a distinct, morphologically continuous outer wall. Received: 3 December 1997 / Accepted: 11 June 1998